Cytoplasmic Damage and Genotoxicity

描述(申请人提供)：自Rventgen发现X射线以来，一个多世纪以来，放射科学的学生一直被教导，辐射引起的遗传损害的典型靶点存在于细胞核的DNA中。细胞质损伤的生物学后果在很大程度上是未知的。使用精确的带电粒子微束和双荧光染料分别定位细胞核和细胞胞浆，从而避免无意中穿越细胞核，申请人先前已经证明，细胞质照射实际上是人-仓鼠杂交(AL)细胞CD59基因的突变，同时造成的细胞毒性最小。在亲本R01的资金支持下，我们最近发现：1)活性氧物种介导这一过程；2)靶向细胞质照射导致膜的脂质过氧化，主要的脂质过氧化副产物4-羟氧基-2-壬烯醛的诱导；3)产生线粒体DNA耗竭(Ao)的AL细胞和人成纤维细胞；最后，靶向细胞质照射通过缝隙连接介导的过程诱导邻近的非辐射细胞之间的旁观者突变和染色单体断裂。这就提出了以下问题：靶向细胞质照射对线粒体DNA突变的影响是什么？线粒体DNA耗尽的人成纤维细胞在细胞质照射时是否表现出诱变减少？这一年补充应用的中心假设是靶向胞浆照射诱导人成纤维细胞线粒体DNA的剂量依赖性突变，以及线粒体DNA耗竭的细胞对遗传毒性信号的反应较差。为了解决这些假设，本文提出了两个具体目标。将对人类成纤维细胞的HPRT基因和线粒体DNA突变(耗尽、异质缺失和点突变)进行评分。拟议的研究将有助于解决细胞质辐射如何导致核内遗传事件的机制。与旁观者诱变效应一起，这项研究将解决一些关于核外靶标和细胞质损伤是如何在哺乳动物细胞中处理的基本问题。 公共卫生相关性：辐射生物学专业的几代学生都被教导，电离辐射引起的可遗传生物效应是辐射-核直接相互作用的结果。利用哥伦比亚大学的带电粒子微束和人类仓鼠杂交(AL)细胞的诱变试验，有证据表明，靶向胞质照射对哺乳动物细胞具有诱变作用。首次明确证明了电离辐射的核外效应，为随后广泛的旁观者/非目标辐射效应提供了强有力的支持。有证据表明，靶向胞质照射也可以引起旁观者效应。因此，更好地了解细胞质辐射的遗传毒性机制在低剂量辐射风险评估中至关重要。线粒体广泛分布于细胞质中，线粒体DNA对氧化损伤非常敏感。这项研究的目的是确定细胞质照射对线粒体DNA突变的可能影响以及随后对线粒体功能的调节。这项研究将解决有关核外靶标以及哺乳动物细胞如何处理细胞质损伤的一些基本问题。

DESCRIPTION (provided by applicant): For over a century since the discovery of X-rays by Rventgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. With the funding support of the parent R01, we have shown recently that 1) reactive oxygen species mediate this process; 2) targeted cytoplasmic irradiation results in lipid peroxidation of membranes as shown by the induction of 4-hdyroxy-2-nonenal, a major lipid peroxidation byproduct; 3) generated mitochondrial DNA depleted (Ao) AL cells and human fibroblasts; and finally, targeted cytoplasmic irradiation induces bystander mutations and chromatid breaks among neighboring, non-irradiate cells through a gap junction-mediated process. This raised the following questions: What are the effects of targeted cytoplasmic irradiation on mitochondrial DNA mutations and does mitochondrial DNA depleted human fibroblasts demonstrated reduced induction of mutagenesis upon cytoplasmic irradiation? The central hypotheses for this one year supplemental application is targeted cytoplasmic irradiation induces a dose dependent mutagenesis of mitochondrial DNA in human fibroblasts and that mitochondrial DNA depleted cells respond poorly to genotoxic signaling. To address these hypotheses, 2 specific aims are proposed. Mutations will be scored at the HPRT locus in human fibroblasts and mitochondrial DNA mutations (depletion, heteroplasmic deletions and point mutations will be determined. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. PUBLIC HEALTH RELEVANCE: Generations of students in radiation biology have been taught that heritable biological effects induced by ionizing radiation are the consequence of a direct radiation-nuclear interaction. Using the Columbia University charged particle microbeam and the human hamster hybrid (AL) cell mutagenic assay, there is evidence that targeted cytoplasmic irradiation is mutagenic in mammalian cells. This first, unequivocal demonstration of an extranuclear effect of ionizing radiation provides strong support of the subsequent, broad reaching bystander/ non-targeted effects of radiation. There is evidence that targeted cytoplasmic irradiation can also induce a bystander effect. As such, a better understanding of the genotoxic mechanism of cytoplasmic irradiation is critical in low dose radiation risk assessment. Mitochondria are widely distributed in the cytosol and mitochondrial DNA is highly susceptible to oxidative damage. The objective of the proposed study is to ascertain the possible effects of cytoplasmic irradiation on mitochondrial DNA mutations and the subsequent modulation on mitochondrial function. The study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.